We propose to isolate and characterize all of the membrane associated enzymes involved in the biosynthesis of the crosslinked peptidoglycan of the bacterial cell wall. This work is based on our finding that it is possible to use cholate and LiC1 to solubilize bacterial membranes capable of peptidoglycan synthesis. The solubilized membranes can be dialysed free of the detergent and high salt concentration to give membranes again capable of peptidoglycan synthesis. Therefore, it is possible to solubilize all of the peptidoglycan synthetic enzymes in reactivatable form. For the enzyme purifications, we propose to use assays based on membrane reconstitution. We have already developed an assay and begun the isolation of a protein which initial evidence suggests is the peptidoglycan polymerase. Our enzyme isolations and characterizations will first be performed with Bacillus megaterium for which we have developed conditions for the solubilization and reconstitution of the peptidoglycan synthetic enzymes. Then we will extend our studies to Escherichia coli and Bacillus subtilis for which good genetic systems are available.